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Biomedical subjects

H Mohler

Publications and source records attributed to H Mohler.

At least 55 records · Page 3Linked to original sources
PubMed1993

GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by alpha 5- and delta-subunit-specific immunopurification.

The pharmacological significance and structural basis of the gamma-aminobutyrate (GABAA) receptor heterogeneity was investigated in situ by an immunobiochemical analysis of receptor populations characterized by the delta- and alpha 5-subunit. Using an antiserum specific for the delta-subunit, a population of GABAA receptors (21 +/- 2% of solubilized receptors) was immunoprecipitated from rat brain extracts which contained high affinity benzodiazepine binding sites. They were distinguished from those immunoprecipitated by the alpha 1- and alpha 3-subunit antisera by a 4-5-fold and 5-10-fold higher affinity for diazepam and beta CCM, respectively. Using the delta-antiserum in immunoaffinity chromatography the delta-subunit was found to be associated with the alpha 1-, alpha 3-, beta 2/3-, and gamma 2-subunits, suggesting that the latter conveys benzodiazepine receptor sensitivity also to GABAA receptors containing the delta-subunit. The receptor population immunoprecipitated by the alpha 5-subunit antiserum (10 +/- 2% of receptors solubilized from whole brain extracts) was characterized by affinities for zolpidem, beta CCM, and CL 218872 which distinguished it from all other known receptor populations. The alpha 5-subunit was associated with the alpha 1-, alpha 3-, beta 2/3- and gamma 2-subunits pointing to differential subunit combinations. Indeed, when receptor populations were immunoprecipitated by the alpha 5-subunit antiserum from different brain regions, zolpidem displayed striking differences in affinity pointing to the role of subunits other than alpha 5 in determining receptor affinity.

Animals↗
PubMed1993

Neuron-specific expression of GABAA-receptor subtypes: differential association of the alpha 1- and alpha 3-subunits with serotonergic and GABAergic neurons.

GABAA-receptors in the brain display a striking structural heterogeneity, which is based on a multiplicity of diverse subunits. The allocation of GABAA-receptor subtypes to identified neurons is essential for an analysis of the functional significance of receptor heterogeneity. Among GABA-receptive neurons, well-characterized examples include the serotonergic and GABAergic neurons in the raphe nuclei. The GABAA-receptor subtypes expressed in these two types of neurons were analysed using antisera which recognize selectively the alpha 1- and alpha 3-subunits, and their co-localization with serotonin and glutamate decarboxylase was assessed by confocal laser microscopy in double and triple immunofluorescence staining in the rat. The vast majority of serotonergic neurons express strong alpha 3-subunit-immunoreactivity, but are devoid of alpha 1-subunit staining. In contrast, both the alpha 1- and alpha 3-subunit-immunoreactivities are present in glutamate decarboxylase-positive neurons. Thus, serotonergic and GABAergic neurons selectively express distinct patterns of alpha subunits, suggesting that they possess distinct subtypes of GABAA-receptors. The occurrence of neuron-specific GABAA-receptor subtypes may open new possibilities for the targeting of drugs with selective therapeutic actions.

Animals↗
PubMed1993

GABAA-receptors: drug binding profile and distribution of receptors containing the alpha 2-subunit in situ.

The highest structural diversity of GABAA-receptor subunits is observed among members of the alpha-subunit class. Using subunit-specific antisera, the receptors containing the alpha 2-subunit were characterized. Western blots revealed an apparent molecular size of 52 kDa for the alpha 2-subunit. Immunohistochemically, the alpha 2-subunit was most preponderant in areas which lack the alpha 1-subunit, e.g. striatum and olfactory bulb granule cell layer, suggesting that these two subunits represent largely distinct receptor subtypes. Pharmacologically, the receptor population which was immunoprecipitated by the alpha 2-subunit-specific antisera displayed a drug binding profile characterized by a low affinity for CL 218872, beta CCM and zolpidem. This is in striking contrast to the high affinities of these ligands displayed by receptors immunoprecipitated by the alpha 1-subunit-specific antiserum. Thus, the alpha 1- and the alpha 2-subunit characterize two GABAA-receptor populations which greatly differ in brain distribution and pharmacological profile.

Amino Acid Sequence↗
PubMed1993

Full and partial agonism displayed by benzodiazepine receptor ligands at recombinant gamma-aminobutyric acidA receptor subtypes.

The differences in intrinsic activity and receptor subtype specificity of the newly developed benzodiazepine receptor ligands bretazenil, divaplon and abecarnil were assessed in recombinant gamma-aminobutyric acidA (GABAA) receptors expressed in mammalian cells from the subunit-cDNA combinations alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2. Chloride currents induced by rapid application of GABA in the presence or absence of drugs were measured using the whole-cell configuration of the patch-clamp technique. Bretazenil displayed an intrinsic activity which amounted only to 58 +/- 7% and 35 +/- 11% of that of flunitrazepam at the alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 combination, respectively. The maximum potentiation by divaplon was only 28 +/- 5% and 21 +/- 9% of that of flunitrazepam at the respective subunit combinations. Thus, the partial agonism postulated for bretazenil and divaplon on pharmacological grounds is shown to be operative on the level of single GABAA receptors. Most strikingly, abecarnil potentiated the GABA response to the same degree as flunitrazepam at the alpha 3 beta 2 gamma 2 combination but to only 52 +/- 14% compared to flunitrazepam at the alpha 5 beta 2 gamma 2 combination. This finding demonstrates that the intrinsic activity of benzodiazepine receptor ligands can vary among the receptor subtypes with the degree of receptor modulation being influenced by the type of alpha subunit.

Benzodiazepinones↗
PubMed1992

GABAA-receptor subtypes differing in alpha-subunit composition display unique pharmacological properties.

GABAA-receptor subtypes in rat brain were characterized using anti-peptide antisera specific for the alpha 1-, alpha 3- and alpha 5-subunits. While a high abundance of alpha 1-containing receptors was demonstrated by immunoprecipitation (80-90% of receptors), the receptors precipitated with the alpha 3- and the alpha 5-antiserum were less frequent (18-25% and 10-23%, respectively). The three receptor populations displayed unique pharmacological properties as shown by radioligand binding. Diazepam, flumazenil and flunitrazepam displayed similar displacing potencies in [3H]-flumazenil binding. However, the affinities of CL 218872, beta CCM and zolpidem were up to 10-fold lower in the alpha 3- than the alpha 1-receptor population while intermediate values were found for the alpha 5-receptor population. In many brain areas, a neuron-specific expression of receptors containing either alpha 1- or alpha 3-subunits could be visualized immunohistochemically. It will be of major interest to determine, whether ligands with differential affinities for receptor subtypes in situ will provide novel therapeutic profiles.

Animals↗
PubMed1991

Identification and immunohistochemical mapping of GABAA receptor subtypes containing the delta-subunit in rat brain.

Synaptic inhibition in brain is mainly mediated via GABAA receptors which display a striking structural heterogeneity. A novel type of GABAA receptor subunit, the delta-subunit, has recently been described based on molecular cloning of its cDNA. To identify the prevalence and distribution of GABAA receptors which contain the delta-subunit protein in situ, polyclonal site-directed antisera were developed against three synthetic peptides derived form the rat delta-subunit cDNA-sequence. All antisera specifically recognized a 54 kDa protein in GABAA receptor preparations. Nearly 30% of the GABAA receptors contained the delta-subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the delta-subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABAA receptors with a delta-subunit have now been visualized and made accessible for a functional analysis of this GABAA receptor subtype in situ.

Amino Acid Sequence↗
PubMed1991

GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.

Recombinant GABAA (gamma-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of alpha-, beta-, and gamma 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Lüddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only alpha- and beta-subunit proteins have so far been detected. To identify the size and distribution of the gamma 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat gamma 2-subunit sequence. On Western blots, both anti-gamma 2-subunit antisera selectively labeled a 43-kDa protein. gamma 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the gamma 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the alpha 1-subunit, whereas bd17 recognizes both the beta 2- and beta 3-subunits (Ewert, M., Shivers, B. D., Lüddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated approximately 90% of the GABAA receptors, the gamma 2-subunit is frequently associated with the alpha 1-subunit and the beta 2- and/or beta 3-subunit in vivo.

Amino Acid Sequence↗
PubMed1991

Immunochemical identification of the alpha 1- and alpha 3-subunits of the GABAA-receptor in rat brain.

To identify subunit variants of the GABAA-receptor antisera were developed against specific cDNA-derived peptide sequences of the alpha 1- and alpha 3-subunits of rat brain. The alpha 1-subunit antiserum selectively recognized a protein of 50 +/- 1 kDa in rat and bovine GABAA-receptor preparations, while the alpha 3-subunit antiserum interacted with a protein doublet of 59 +/- 2 kDa and 61 +/- 3 kDa. The alpha 1-subunit immunoreactivity resides in a large population of GABAA-receptors as shown by immunoprecipitation of 63 +/- 6% of [3H]flumazenil binding sites with the alpha 1-subunit antiserum. In contrast, only 24 +/- 3% of receptor binding sites were precipitated with the alpha 3-subunit antiserum. Co-precipitation studies suggest that the alpha 1- and alpha 3-subunit immunoreactivities do not share the same receptor population while the gamma 2-subunit immunoreactivity is associated with the alpha 1-subunit immunoreactivity.

Amino Acid Sequence↗
PubMed1990

Identification of the gamma 2-subunit protein in native GABAA receptors in brain.

GABAA receptors with functional benzodiazepine receptors have been reconstituted by coexpression of alpha-, beta- and gamma-subunit cDNAs. In brain, proteins of the alpha- and beta-subunits, but not the gamma 2-subunit have been identified. Using an antipeptide antiserum, we now demonstrate that the gamma 2-subunit is a protein of 43 kDa. It is present in at least 50% of GABAA receptors, as shown by immunoprecipitation.

Amino Acid Sequence↗
PubMed1990

GABAA-receptors: structural requirements and sites of gene expression in mammalian brain.

GABAA-receptors, the major synaptic targets for the neurotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABAA-receptors are still elusive. Expression of cDNAs coding for the alpha 1- beta 1-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABAA-receptors with fully functional benzodiazepine receptor sites were formed when the alpha 1- and beta 1-subunits were co-expressed with the gamma 2-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, beta 1, beta 2, beta 3, gamma 2) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the alpha 1-, beta 1- and gamma 2-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABAA-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.

Animals↗
PubMed1990

GABA, GABA receptors and benzodiazepine receptors in the human spinal cord: an autoradiographic and immunohistochemical study at the light and electron microscopic levels.

The regional, cellular and subcellular distribution of GABA, GABA receptors and benzodiazepine receptors was investigated by light and electron microscopy in the human lumbar spinal cord taken post-mortem from eight cases aged 20-76 years. Firstly, the regional distribution of GABA receptors and benzodiazepine receptors was studied using autoradiography following in vitro labelling of cryostat sections with tritiated ligands. This was followed by a detailed study of the cellular and subcellular distribution and localization of GABA and benzodiazepine/GABAA receptors by light and electron microscopy using immunohistochemical techniques with monoclonal antibodies to GABA and to the alpha and beta subunits of the benzodiazepine/GABAA receptor complex. The results showed a close correspondence in the regional distributions of GABA, GABA (GABAA and GABAB) receptors and benzodiazepine receptors. The highest density of GABA-like immunoreactivity, GABA receptors and benzodiazepine receptors was localized as a dense band within lamina II of the dorsal horn (especially inner lamina II) with moderately high densities in laminae I and III. The remaining laminae of the spinal gray matter showed much lower levels of labelling. A close correspondence was also seen in the distribution of GABA-like immunoreactivity and of benzodiazepine/GABAA receptor immunoreactivity at the cellular and subcellular levels. At the cellular level, the greatest number of GABA-immunoreactive cells was found in lamina II; they comprised small, round to oval cells and, on the basis of soma size, shape, orientation and dendromorphology, they corresponded to previously described islet and filamentous cells. Benzodiazepine/GABAA receptor immunoreactivity was also localized on the same cell types in lamina II. At the subcellular level in lamina II, GABA-immunoreactive axon terminals mainly established axodendritic synaptic contacts. Small numbers of GABA-immunoreactive axon terminals appear to form possible axo-axonic contacts in complex synaptic arrays. Benzodiazepine/GABAA receptors were localized within the same types of synaptic complexes in which GABA-immunoreactive axon terminals were found. In these synaptic complexes, benzodiazepine/GABAA receptor immunoreactivity was associated with presynaptic and postsynaptic membranes and on apparent non-synaptic membranes. These results show a high concentration of GABA, GABA receptors and benzodiazepine receptors in lamina II of the dorsal horn of the human spinal cord and suggest a possible role for GABA in spinal sensory functions.

Autoradiography↗
PubMed1990

Synaptic and Nonsynaptic Localization of Benzodiazepine/GABAA Receptor/Cl- Channel Complex Using Monoclonal Antibodies in the Dorsal Lateral Geniculate Nucleus of the Cat.

The two monoclonal antibodies, bd-17 and bd-24, are specific for beta- and alpha-subunits of the GABAA/benzodiazepine receptor/chloride channel complex respectively. An abundance of both subunits has been revealed in the visual thalamus of the cat by light microscopic immunocytochemistry using these antibodies. The alpha-subunit specific antibody and electron microscopy were used to determine the subcellular distribution of immunoreactivity with respect to specific cell classes in the dorsal lateral geniculate nucleus. Immunoreactivity was always associated with membranes and the degree of immunoreactivity varied greatly between different types of cell as defined by: (i) immunoreactivity for GABA; (ii) soma area; (iii) presence or absence of cytoplasmic laminated bodies (CLB). GABA negative neurons with the smallest soma area showed the strongest immunoreactivity, mainly in the endoplasmic reticulum and also on the somatic plasma membrane. Cytoplasmic laminated bodies could be found in the majority of these neurons. Large GABA negative cells without CLBs were strongly immunoreactive on the plasma membrane of the soma and dendrites, but showed scant if any intracellular immunoreactivity. GABA-positive cells showed weak intracellular immunoreactivity but negligible if any immunoreactivity at the somatic and proximal dendritic plasma membrane. A similar reaction pattern was found in GABA negative cells which contained no CLBs and which constituted a medium sized cell population. It is suggested that the degree of intracellular receptor immunoreactivity is positively correlated with receptor turnover. The dendrites of projection cells, particularly outside the glomeruli, showed strong immunoreactivity on the plasma membrane. The synaptic junctions formed by many boutons (F terminals) establishing symmetrical synapses with dendrites of relay cells were immunopositive, but no immunoreactivity could be detected at the synapses established by the presynaptic dendrites of the local interneurons. Many axo-somatic F1 junctions were also immunoreactive. However, immunoreactivity for the receptor/channel complex was also widely distribution on nonsynaptic plasma membranes of somata and dendrites. Thus GABA may act at both synaptic and non-synaptic sites. Furthermore, the correlation of immunoreactivity for the GABAA receptor complex with previously published properties of physiologically identified cells suggests that the strongly immunoreactive, small, GABA negative cells with CLBs might correspond to the 'lagged' X-type cells, and the large GABA negative receptor outlined cells without CLBs might correspond to some of the Y-type neurons.

Journal Article↗
PubMed1990

Functional characteristics and sites of gene expression of the alpha 1, beta 1, gamma 2-isoform of the rat GABAA receptor.

GABAA receptors, the major synaptic targets for the neurotransmitter GABA, constitute gated chloride channels. By their allosteric, drug-induced modulation, they serve as control elements for the regulation of anxiety, vigilance, and epileptiform activity. The structural requirements of fully functional GABAA receptors in the mammalian brain have remained elusive so far. We report here on the cloning of the gamma 2-subunit cDNA of rat brain and its functional analysis by coexpression with the alpha 1- and beta 1-subunits in Xenopus oocytes, and on the sites of gene expression of the 3 subunits in the rat brain. The recombinant receptor displayed GABA-inducible currents (Imax = 6 microA; Ka = 75 microM) which were allosterically modulated by benzodiazepine receptor ligands (enhancement and inhibition by diazepam and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, respectively). In the absence of GABA, pentobarbital elicited a maximal current amplitude similar to that of GABA. A minor population of channels is expressed which is open in the absence of GABA or pentobarbital. Mapping subunit gene expression by in situ hybridization histochemistry suggests that the alpha 1-, beta 1-, and gamma 2-subunits are likely receptor constituents in some neuronal populations, e.g., mitral cells of the olfactory bulb, pyramidal cells of the hippocampus, and granule cells of the dentate gyrus and cerebellum.

Amino Acid Sequence↗
PubMed1989

Subcellular localization of benzodiazepine/GABAA receptors in the cerebellum of rat, cat, and monkey using monoclonal antibodies.

Two monoclonal antibodies, bd-17 and bd-24, specific for the beta- and alpha-subunit of the GABAA/benzodiazepine receptor/chloride channel complex, respectively, were used to determine the subcellular distribution of immunoreactivity in the cerebellum by electron microscopy. The 2 antibodies showed similar antigen distribution on the plasma membrane (except in the rat; bd-24 does not recognize the rat antigen), but intracellular immunoreactivity was more prevalent for the alpha-subunit. The plasma membrane of all neuronal types was immunopositive. The degree of immunoreactivity varied greatly between different types of cell, but it was stereotyped among individual cells of the same type. Granule cells showed the strongest immunoreactivity, not only on their dendrites which receive synapses from GABA-containing Golgi cell terminals, but also on their somata which do not receive synapses. Stellate and basket cells were somewhat weaker in immunoreactivity. Purkinje cells were only weakly positive on their somatic membrane but stronger on their dendritic shafts and spines. Golgi cells showed negligible if any immunoreactivity. Neurons of the deep cerebellar nuclei were strongly immunopositive along their plasma membrane. Immunoreactivity was strong in cisternae of the endoplasmic reticulum and in the Golgi saccules of stellate and basket cells, variable in Purkinje cells, while granule cells were rarely immunoreactive intracellularly. It is suggested that these differences reflect differences in the turnover of the receptor complex in the different cell types. The synaptic clefts established by boutons of the GABAergic stellate, basket, and Golgi cells were immunopositive, as were many synapses in the deep cerebellar nuclei. However, immunoreactivity was also present along the nonjunctional plasma membrane, and it was concluded that this reflected the distribution of the antigen. The synaptic clefts at the presumed glutamate-releasing parallel and mossy fiber terminals were almost always immunonegative. No immunoreactivity was detected on axons, nerve terminals, or glial cells. The results demonstrate that different neuronal types express the GABAA/benzodiazepine receptor/chloride channel complex to different degrees. The distribution of the receptor complex suggests that the cellular topography of GABAergic influence is not governed by the precise spatial arrangement of the receptors but by the precise placement of the GABA-releasing terminals, a characteristic of the cerebellar circuit.

Animals↗